During the past few years, the mechanisms by which electron transfer chains generate proton gradients for coupling to ATP synthesis have been established in broad outline for a number of systems. The quinol oxidizing complexes of respiration and photosynthesis all act through Q-cycle mechanisms, and catalyse oxidation of a quinol, reduction of two equivalents of cytochrome c (or an equivalent protein), the transfer of two H+/2e- across the membrane, and the release of two additional protons to the aqueous phase containing cytochrome c. We propose to use a combination of techniques to study the three catalytic sites of the ubiquinol:cytochrome c2 oxidoreductase of Rps. sphaeroides. In this photosynthetic bacterium, turn-over of the complex can be easily initiated by flashes of light, and kinetic and thermodynamic approaches have been used to characterise the mechanism in some detail. The catalytic sites identified are a ubiquinol oxidase site, a quinone (semiquinone) reductase site, and a cytochrome c2 reductase site. Similar kinetic methods can be used to study the complex in which catalytic sites have been modified by mutation. By studying mutant strains in which the lesion has been located at a specific catalytic site, we propose to characterise the peptide sequences contributing to particular sites. We will then use site directed mutagenesis to specifically modify the amino acids at the catalytic site, and analyse the consequent effects on catalysis. We will complement these studies with an investigation of the topography of the complex in the membrane, using antibodies directed against specific hydrophilic segments of the polypeptide chain. We expect to be able to map the detailed architecture and topology of the catalytic sites, and to show how specific groups contribute to the catalytic mechanism.
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